The long-term objective of these studies is to define the physiological differences between the cellular Ca2+ homeostasis of normal and neoplastic tissues. The rationale for this project is based on observations that tumor cells exhibit abnormal growth characteristics with respect to extracellular Ca2+ and that their maintenance and regulaton of intracellular Ca2+ may also be altered. The specific aims of this research are: (1)\to determine the cytosolic-free Ca2+ con-centration and the intracellular distribution of Ca2+ in hepatocytes (isolated from normal and regenerating liver) as well as malignant hepatoma cells; (2)\to determine the Ca2+ buffering characteristics of normal liver and hepatoma mitochondria and endoplasmic reticulum under physiologically realistic conditions; and (3)\to compare the effects of Ca2+ on the cytoskeleton of normal and malignant tissues. Recent progress along these lines include: (1)\development of a substantially improved technique for isolating mitochondria from ascites tumor cells and comparingtheir respiratory characteristics with those of digitonin-permeabilized cells (Anal. Biochem. 137,360-367, 1984); (2)\demonstration that AS30-D hepatoma mitochondria do not release Ca2+ as normal liver mitochondria do in response to the presence of t-butyl hydroperoxide and that this is due to abnormally low levels of glutathione peroxidase and reductase activities in the tumor mito-chondria (Fed. Proceed. 43, 2003, 1984); (3)\demonstration that AS30-D hepatoma mitochondria buffer the free Ca2+ concentration of a cytosol-like medium at a significantly higher level than normal liver mitochondria do and that this is due to an abnormally high rate of Ca2+ efflux; and(4)\preliminary indications that microsomal vesicles from AS30-D hepatoma cells accumulate more Ca2+ at a faster rate than that observed with normal liver microsomes. (E)